Although lumen generation has been extensively studied through so-called cyst-formation assays in Madin-Darby canine kidney (MDCK) cells, an underlying mechanism that leads to the initial appearance of a solitary lumen remains elusive. Lumen formation is thought to take place at early stages in aggregates containing only a few cells. Evolutionarily conserved polarity protein complexes, namely the Crumbs, Par, and Scribble complexes, establish apicobasal polarity in epithelial cells, and interference with their function impairs the regulated formation of solitary epithelial lumina. Here, we demonstrate that MDCK cells form solitary lumina during their first cell division. Before mitosis, Crumbs3a becomes internalized and concentrated in Rab11-positive recycling endosomes. These compartments become partitioned in both daughter cells and are delivered to the site of cytokinesis, thus forming the first apical membrane, which will eventually form a lumen. Endosome trafficking in this context appears to depend on the mitotic spindle apparatus and midzone microtubules. Furthermore, we show that this early lumen formation is regulated by the apical polarity complexes because Crumbs3 assists in the recruitment of aPKC to the forming apical membrane and interference with their function can lead to the formation of a no-lumen or multiple-lumen phenotype at the two-cell stage.

Establishment of epithelial apicobasal polarity is crucial for proper kidney development and function. In recent years, there have been important advances in our understanding of the factors that mediate the initiation of apicobasal polarization. Key among these are the polarity complexes that are evolutionarily conserved from simple organisms to humans. Three of these complexes are discussed in this review: the Crumbs complex, the Par complex, and the Scribble complex. The apical Crumbs complex consists of three proteins, Crumbs, PALS1, and PATJ, whereas the apical Par complex consists of Par-3, Par-6, and atypical protein kinase C. The lateral Scribble complex consists of Scribble, discs large, and lethal giant larvae. These complexes modulate kinase and small G protein activity such that the apical and basolateral complexes signal antagonistically, leading to the segregation of the apical and basolateral membranes. The polarity complexes also serve as scaffolds to direct and retain proteins at the apical membrane, the basolateral membrane, or the intervening tight junction. There is plasticity in apicobasal polarity, and this is best seen in the processes of epithelial-to-mesenchymal transition and the converse mesenchymal-to-epithelial transition. These transitions are important in kidney disease as well as kidney development, and modulation of the polarity complexes are critical for these transitions.

Mammalian Lin-7 forms a complex with several proteins, including PALS1, that have a role in polarity determination in epithelial cells. In this study we have found that loss of Lin-7 protein from the polarized epithelial cell line Madin-Darby canine kidney II by small hairpin RNA results in defects in tight junction formation as indicated by lowered transepithelial electrical resistance and mislocalization of the tight junction protein ZO-1 after calcium switch. The knock down of Lin-7 also resulted in the loss of expression of several Lin-7 binding partners, including PALS1 and the polarity protein PATJ. The effects of Lin-7 knock down were rescued by the exogenous expression of murine Lin-7 constructs that contained the L27 domain, but not the PDZ domain alone. Furthermore, exogenously expressed PALS1, but not other Lin-7 binding partners, also rescued the effects of Lin-7 knock down, including the restoration of PATJ protein in rescued cell lines. Finally, the effects of Lin-7 knock down appeared to be due to instability of PALS1 protein in the absence of Lin-7, as indicated by an increased rate of PALS1 protein degradation. Taken together, these results indicate that Lin-7 functions in tight junction formation by stabilizing its membrane-associated guanylate kinase binding partner PALS1.Polarization of mammalian epithelia is characterized by the asymmetrical partitioning of proteins to specific membrane domains. The cellular boundary of the apical and basolateral membranes in polarized epithelia is demarcated by the tight junction that forms a fence between the two membrane compartments. Junctions within and between cells are defined by the localization of, and interactions between, specific protein complexes. The interaction of these proteins is mediated by evolutionarily conserved protein-protein interaction domains. Many studies have shown an important role for PDS-95/Discs large/ZO-1 (PDZ) domain-containing proteins in tight junctions and the polarized localization of proteins (1, 2

Summary Disruption of the primary cilium is associated with a growing number of human diseases collectively termed ciliopathies. Ciliopathies present with a broad range of clinical features consistent with the near ubiquitous nature of the organelle and its role in diverse signaling pathways throughout development and adult homeostasis. The clinical features associated with cilia dysfunction can include such phenotypes as polycystic kidneys, skeletal abnormalities, blindness, anosmia, and obesity. Although the clinical relevance of the primary cilium is evident, the effects that cilia dysfunction has on the cell and how this contributes to disease remains poorly understood. Here, we show that loss of ciliogenesis genes such as Ift88 and Kif3a lead to increases in post-translational modifications on cytosolic microtubules. This effect was observed in cilia mutant kidney cells grown in vitro and in vivo in cystic kidneys. The hyper-acetylation of microtubules resulting from cilia loss is associated with both altered microtubule stability and increased α-tubulin acetyl-transferase activity. Intriguingly, the effect on microtubules was also evident in renal samples from patients with autosomal recessive polycystic kidneys. These findings indicate that altered microtubule post-translational modifications may influence some of the phenotypes observed in ciliopathies.

During Epithelial to Mesenchymal Transition (EMT), cells modulate expression of proteins resulting in loss of apical-basal polarity. Effectors of this EMT switch target the polarity protein Crumbs3a, a small transmembrane protein that is essential for generation of the apical membrane and tight junctions of mammalian epithelial cells. We previously showed that the Crumbs3 gene is a direct target of transcriptional regulation by Snail, a potent inducer of EMT. However, Snail has also been shown to have multiple non-transcriptional roles, including regulation of cell adhesion, proliferation and survival. Using SNAP-tag labeling, we determined that cell surface Crumbs3a has a half-life of approximately 3 hours and that this cell surface half-life is significantly reduced when EMT is induced by Snail. We further observe that Snail induces differential glycosylation of Crumbs3a, including sialylation, suggesting a mechanism by which Crumbs3a may be destabilized. These results indicate that Crumbs3a is a post-translational target of Snail, in addition to being a transcriptional target. We conclude that Snail’s ability to post-translationally modify and destabilize Crumbs3a augments the depolarizing process of EMT.